Jennifer A. Waters

The identification of tumor-initiating cells (TICs) has traditionally relied on surface markers including CD133, CD44, CD117, and the aldehyde dehydrogenase (ALDH) enzyme, which have diverse expression across samples. A more reliable indication of TICs may include the expression of embryonic transcription factors that support long-term self-renewal, multipotency, and quiescence. We hypothesize that SOX2, OCT4, and NANOG will be enriched in ovarian TICs and may indicate TICs with high relapse potential. We evaluated a panel of eight ovarian cancer cell lines grown in standard 2-D culture or in spheroid-enriching 3-D culture, and correlated expression with growth characteristics, TIC marker expression, and chemotherapy resistance. RNA-sequencing showed that cell cycle regulation pathways involving SOX2 were elevated in 3-D conditions. HGSOC lines had longer doubling-times, greater chemoresistance, and significantly increased expression of SOX2, OCT4, and NANOG in 3-D conditions. CD117+ or ALDH+/CD133+ cells had increased SOX2, OCT4, and NANOG expression. Limiting dilution in in vivo experiments implicated SOX2, but not OCT4 or NANOG, with early tumor-initiation. An analysis of patient data suggested a stronger role for SOX2, relative to OCT4 or NANOG, for tumor relapse potential. Overall, our findings suggest that SOX2 may be a more consistent indicator of ovarian TICs that contribute to tumor repopulation following chemotherapy. Future studies evaluating SOX2 in TIC biology will increase our understanding of the mechanisms that drive ovarian cancer relapse.

Identification of tumor initiating cells (TICs) has traditionally relied on expression of surface markers such as CD133, CD44, and CD117 and enzymes such as aldehyde dehydrogenase (ALDH). Unfortunately, these markers are often cell type specific and not reproducible across patient samples. A more reliable indication of TICs may include elevated expression of stem cell transcription factors such as SOX2, OCT4, and NANOG that function to support long-term self-renewal, multipotency, and quiescence. RNA-sequencing studies presented here highlight a potential role for SOX2 in cell cycle progression in cells grown as 3-D spheroids, which are more tumorigenic and contain higher numbers of TICs than their 2-D monolayer cultured counterparts. SOX2, OCT4, and NANOG have not been comprehensively evaluated in ovarian cancer cell lines, although their expression is often associated with tumorigenic cells. We hypothesize that SOX2, OCT4, and NANOG will be enriched in ovarian TICs and will correlate with chemotherapy resistance, tumor initiation, and expression of traditional TIC markers. To investigate this hypothesis, we evaluated SOX2, OCT4, and NANOG in a panel of eight ovarian cancer cell lines grown as a monolayer in standard 2-D culture or as spheroids in TIC-enriching 3-D culture. Our data show that the high-grade serous ovarian cancer (HGSOC) lines CAOV3, CAOV4, OVCAR4, and OVCAR8 had longer doubling-times, greater resistance to chemotherapies, and significantly increased expression of SOX2, OCT4, and NANOG in TIC-enriching 3-D culture conditions. We also found that in vitro chemotherapy treatment enriches for cells with significantly higher expression of SOX2. We further show that the traditional TIC marker, CD117 identifies ovarian cancer cells with enhanced SOX2, OCT4, and NANOG expression. Tumor-initiation studies and analysis of The Cancer Genome Atlas (TCGA) suggest a stronger role for SOX2 in ovarian cancer relapse compared with OCT4 or NANOG. Overall, our study clarifies the expression of SOX2, OCT4, and NANOG in TICs from a variety of ovarian cancer cell lines. Our findings suggest that SOX2 expression is a stronger indicator of ovarian TICs with enhanced tumor-initiation capacity and potential for relapse. Improved identification of ovarian TICs will advance our understanding of TIC biology and facilitate the design of better therapies to eliminate TICs and overcome chemotherapy resistance and disease relapse.

High Grade Serous Ovarian Cancer (HGSOC) is the most lethal gynecological disease in women, with over 70% of advanced stage disease relapsing within 18 months. Recent studies suggest that tumor-initiating cells (TICs) support tumor heterogeneity, chemoresistance, and relapse in HGSOC. Identification of TICs has traditionally relied on expression of surface markers such as CD133, CD44, and CD117 and/or enzymes such as aldehyde dehydrogenase (ALDH). Unfortunately, these markers are often cell type specific and not reproducible across patient samples. More reliable markers of TICs may include stem cell transcription factors such as Sox2, Oct4, and Nanog that function to support long-term self-renewal, multipotency, and quiescence, although these factors have not been comprehensively evaluated in ovarian cancer. We have previously shown that ovarian cancer cells cultured in 3D spheroid-supporting conditions have enhanced expression of Sox2, Oct4, and Nanog and are more tumorigenic than cells cultured in 2D as a monolayer. We hypothesize that Sox2, Oct4, and Nanog support spheroid formation, chemotherapy resistance, and tumorigenicity more reliably than traditional TIC markers. To investigate this hypothesis we evaluated five HGSOC cell lines (CAOV3/CAOV4/OV90/OVCAR4/OVCAR8), two undefined serous lines (ACI23, OVCAR5) and one non-serous line (SKOV3) in 2D vs 3D conditions and quantitated Sox2, Oct4, and Nanog expression, spheroid formation, chemotherapy resistance and tumorigenicity. Our data show that OVCAR4, OVCAR5, and OVCAR8 had the highest expression of Sox2, Oct4, and Nanog in 3D conditions and at least half of the cell lines had enriched expression of all three factors after chemotherapy treatment. A correlation analysis of the traditional TIC markers shows that the surface marker CD133 positively correlated with expression of Sox2, Oct4, and Nanog. Analysis of TCGA data shows that Sox2, Oct4, and Nanog are prone to amplification or gain of function events in 21%, 4%, and 7% of patients, respectively, and these patients are significantly more likely to have recurrent disease. Overall our findings suggest that Sox2, Oct4, and Nanog correlate with TIC features and may be more reliable markers for the identification of cells with enhanced tumor-initiation capacity and drug resistance. Our data further clarify specific ovarian cancer cell lines that have greater dependence on these factors. More reliable and consistent identification of ovarian TICs will improve our understanding of TIC biology and enable the design of therapies to overcome chemotherapy resistance and disease relapse. Citation Format: Mikella Robinson, Samuel F. Gilbert, Logan J. Alexander, Samuel E. Green, Omar Lujano-Olazaba, Omid Patrus, Jennifer A. Waters, Jacqueline M. Lara, Carrie D. House. Sox2, Oct4, and Nanog expression for identification of ovarian cancer tumor initiating cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4960.

Background: Ovarian cancer is the most lethal gynecologic malignancy mainly due to a high prevalence of chemotherapy-resistant recurrent disease. This mechanism is unknown but recent studies have pointed to a small population of chemoresistant cancer stem cells (CSC) as the driver of these recurrences. Traditional surface markers of cancer stem cells (CD44, ALDH, CD117, etc.) have shown mixed success in identifying this subpopulation, with different cell lines and patient samples exhibiting dissimilar profiles. These markers are often related to the cell of origin and are not necessarily specific to CSCs. As such, a reporter that responds to functional features of stem cells would be beneficial in identifying the CSC subpopulation regardless of cell line and patient sample. Here we demonstrate that using a reporter to detect SOX2 and OCT4 overexpression (called SORE6) can identify the CSC subpopulation within ovarian cancer cell lines. Methods: The SORE6 reporter was stably transduced into OV90 and ACI23 ovarian cancer cell lines. The subpopulation of cells having high SOX2/OCT4 expression (SORE6+) were isolated using fluorescence assisted cell sorting (FACs) and evaluated for CSC characteristics relative to SORE6- cells. Gene expression, viability, spheroid formation capacity, and tumorigenicity was assessed for both SORE6+ and SORE6- cells. In vivo limiting dilution analyses were performed on athymic nude female mice using subcutaneous injection. Results: Compared to the bulk population which has low levels of SOX2/OCT4 expression (SORE6-), SORE6+ cells showed a 1.5-fold increase in SOX2/OCT4/NANOG transcript levels in ACI23 cells and 2 to 15-fold increase in these transcript levels in the OV90 cells. SORE6+ cells showed enhanced chemoresistance when treated with conventional chemotherapies for ovarian cancer, carboplatin and paclitaxel, although this difference was more dramatic in the OV90 cells (four-fold) relative to ACI23 (two-fold). These findings indicate that SOX2/OCT4 expression could contribute to drug resistance and clinical recurrence. SORE6+ cells also showed an increased capacity to form spheroids in low-serum conditions and again this difference was more dramatic in the OV90 cells. In vivo limiting dilution studies are underway but appear to show significant differences in tumorigenicity at low dilutions of ≤5,000 cells/mouse. Conclusion: The SORE6 reporter construct identifies CSC populations within ovarian cancer cell lines and may be a reliable tool for isolating CSCs that depend on SOX2 and OCT4 expression. Use of this reporter will enhance our understanding of mechanisms that support chemoresistance and enable the study of CSC dynamics within the tumor microenvironment, both of which have significant implications for ovarian cancer progression and treatment. Citation Format: Samuel F. Gilbert, Mikella Robinson, Logan A. Alexander, Omar Lujano-Olazaba, Jacqueline M. Lara, Jennifer A. Waters, Omid Patrus, Alex Horkowitz, Carrie D. House. Identification and isolation of ovarian cancer stem cell populations using a novel functional reporter may provide new insights into ovarian cancer cell dynamics and drug resistance [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4941.

Ovarian cancer is the most lethal gynecological malignancy in the United States, with a known predilection for metastasizing to the omentum, a sheet of fatty peritoneal tissue that encloses the abdomen. This finding coincides with some evidence that ovarian cancer is more aggressive in the obese setting with worse overall survival. While it has been shown that ovarian cancer cells invade towards adipocytes in vitro, the mechanism by which they support formation of secondary tumors is not well understood. We hypothesize that soluble factors enriched in the obese setting are activating signaling pathways in tumor cells to drive migration and proliferation. To evaluate this hypothesis we co-cultured primary human omental adipocytes derived from obese female donors with ovarian cancer cell lines and profiled secretory factors (cytokines and adipokines) responsible for increasing tumor initiation in vivo and clonogenicity, proliferation, and drug resistance in vitro. Secretion of cytokines IL-6, IL-8, and CCL2 by adipocytes is enhanced when co-cultured with ovarian cancer cells. A subcutaneous tumor model showed that low dilutions of ovarian cancer cells require adipocytes for engraftment and tumor formation. A transplantation assay showed that ovarian cancer cells require the presence of adipocytes for sustained tumor-initiation capacity. Studies are underway to investigate adipocyte-secreted factors in this process. Our findings highlight the role of adipocytes in the ovarian tumor microenvironment and uncover signaling factors that may enhance ovarian cancer progression. Understanding these processes will enable the design of more effective therapies for treating ovarian cancer. Citation Format: Jennifer Waters, Mikella Robinson, Samuel F. Gilbert, Logan J. Alexander, Carrie D. House. Role of secretory factors from obese-derived omentum in supporting ovarian cancer progression [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6116.